Porphobilinogenase from Rhodopseudomonas palustris

Comp. Biochem. Physiol. Vol. 92B, No. 2, pp. 291-295, 1989

0305-0491/89 $3.1)0+ 0.00 Pergamon Press pie

Printed in Great Britain

PORPHOBILINOGENASE FROM RHODOPSEUDOMONAS PALUSTRIS* A. A. JUKNAT,M. L. KOTLER,G. E. KOOPMANN and A. M. DEL C. BATLLEt Centro de Investigaciones sobre Porfirinas y Porfirias, CIPYP (FCEN, UBA--CONICET), Ciudad Universitaria, Pabell6n II, 2do. Piso, 1428 Buenos Aires, Argentina

(Receded 15 March 1988) Rp. palustris has been isolated and some properties of a partially purifed fraction were studied. 2. PBGase has an optimum pH of 7.4 when activity was expressed in terms of porphyrins formed and two pH maxima at 7.4 and 8.5 when activity was based on the amount of PBG consumed. 3. Cyclotetramerization rate and distribution of reaction products were not affected either by the presence or absence of oxygen. 4. Two PBGase active species of mol. wt 115,000 and 50,000 were found, by means of gel filtration through a calibrated Sephadex G-100 column. 5. Kinetic data show the existence of positive cooperative effects for porphyrin formation, while a hyperbolic behaviour for PBG consumption was observed. A b s t r a c t - - l . Porphobilinogenase (PBGase) from

INTRODUCTION Uroporphyrinogen I synthetase (URO-S), also known as PBG-deaminase (EC 4.3.1.8) catalyzes the head to tail condensation of four molecules of PBG to form the tetrapyrrole intermediate hydroxymethylbilane (Battersby et al., 1979a; Burton et al., 1979) which in the presence of uroporphyrinogen III synthase (isomerase, cosynthetase, EC 4.2.1.75) is converted into uroporphyrinogen III (urogen III) (Battersby et al., 1979a, b, 1982, 1983; Jordan et al., 1979). The enzyme complex deaminase-isomerase is usually called porphobilinogenase (PBGase) as early suggested by Lockwood and Rimington (1957). In the absence of cosynthetase, the hydroxymethylbilane spontaneously cyclizes to form uroporphyrinogen I (urogen I). A great deal of information about the isolation, purification and properties of PBGase from animal sources, cultured cells and Euglena gracilis has been accumulated in our laboratory (Sancovich et al., 1969; Rossetti et al., 1986, and Refs therein, 1987). One of the earliest studies on porphyrin biosynthesis from PBG was carried out in Rhodopseudomonas spheroides (Heath and Hoare, 1959; Hoare and Heath, 1959). Purification and properties of U R O - S from this bacteria (Davies and Neuberger, 1973; Jordan and Shemin, 1973) and studies on possible intermediates (Davies and Neuberger, 1973) were also reported. In view of the scarce information existing in photosynthetic bacteria, we have chosen one member of the family Rhodospirillaceae as a good and interesting source for our studies on

*Dedicated to Professor Claude Rimington FRS, on the occasion of his 85th birthday. tAll correspondence should be addressed to: Professor Alcira Batlle, Viamonte 1881-10 "A", 1056 Buenos Aires, Argentina. 291

PBGase. The present paper will therefore describe the isolation and some interesting properties of a partially purified PBGase fraction obtained from Rhodopseudomonas palustris, the most c o m m o n nonsulphur purple bacteria. MATERIALS AND METHODS

Rhodopseudomonas palustris (wild type) was grown semianaerobically in the medium of Cohen-Bazire et al. (1957) containing 0.1% peptone and 0.1% yeast extract. The bacteria was incubated and harvested as described by Kotler et al. (1987a). All procedures from this step onwards were performed at 4°C. Sephadex gels were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden and calcium phosphate gel was prepared according to Keilin and Hartree (1951). Other methods or materials not specified here were those already indicated by Kotler et al. (1987a). Partial purification of Rp. palustris PBGase Frozen bacteria were suspended in 0.05 M sodium phosphate buffer, pH 7.4, up to 60 mg/ml and disrupted by ultrasonic treatment for 60sec in an ultrasonic power unit (Heat Systems Ultrasonics, W 185 D). The resulting fraction was diluted with the same buffer (1:3, v/v) and the homogenate centrifuged at 27,000g for 1 hr. The resulting supernatant was treated with ammonium sulphate, the fraction precipitating at 35-55% saturation was collected and dissolved in a small volume of 0.05 M Tris-HC1 buffer, pH 7.4, and desalted through a Sephadex G-25 column (1.8 x 50cm). The eluate was treated with calcium phosphate gel (2 mg gel/mg protein) and the supernatants containing the active enzyme were collected and concentrated by adding ammonium sulphate to 55% saturation. The protein precipitate dissolved in a minimal volume of the same buffer was applied to a Sephadex G-100 column (2.5 × 57 cm), equilibrated and eluted with 50 mM Tris-HCl buffer, pH 7.4. Fractions containing enzyme activity were combined and concentrated by ammonium sulphate precipitation (0-55%). The enzyme was purified about 22-fold with a very high specific activity (161nmol urogen III/2 hr/mg protein).

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Determination of enzyme actieity The standard reaction mixture contained, in a final volume of 1.5 ml, 0.05 M Tris-HC1 buffer, pH 7.4, enzyme fraction (usually 0.4 mg protein) and 60 #g PBG prepared in the same buffer. Incubation was carried out aerobically in the dark for 2 hr at 37°C with mechanical shaking. Blanks were always run with PBG and without enzyme. The reaction was stopped by the addition of 10% TCA (final concentration 5% w/v) and the tubes were exposed to light for 20 min to oxidize the porphyrinogens to porphyrins. After removing the precipitated protein, porphyrins formed and remaining PBG were estimated in the resulting supernatant solution (Rimington, 1960: Moore and Labbe, 1964). Isolation, identification, quantitative determination and isomeric composition of the different porphyrins formed were carried out as previously indicated by Kotler et al. (1987a). One unit of PBGase was defined as the amount of enzyme that catalyzes the formation of 1 nmol of uroporphyrinogen 111/2 hr in the standard assay system; specific activity being units/mg protein. Protein concentration was determined by the methods of Lowry et al. (1951) and Bradford (1976).

Molecular wt determination Molecular wt was estimated as described by Baffle (1967), employing a calibrated Sephadex G-100 column (2.5 x 57 cm), eluted with 0.05 M Tris-HC1 buffer pH 7.4. RESULTS

AND

DISCUSSION

Bacterial growth The density of Rp. palustris cultures was followed during cell growth by turbidimetric measurements reading the absorption at 680 nm. Starting with an inoculum of cells in the stationary period, the appearance of the three classic phases of bacterial growth was observed (Fig. 1). When enzyme activity was measured, a linear but slow increase up to 7 0 h r was found, followed by a more rapid enhancement occurring during the stationary phase. Rp. palustris cells were then harvested after 72 hr of g r o w t h - - i n the beginning of the last phase and before essential nutrients become limited or inhibitory metabolic products accumulate in the incubation medium.

Isolation conditions A m o n g the variables tested to determine the optimum isolation conditions, buffer, pH and sonication

time were investigated. The best solution lbr extracting the enzyme was found to be either 0.05 M sodium phosphate or 0.05 M Tris-HC1 buffer at pH range 7.4-7.8. Concentrated cell suspensions (60mg/ml) were disrupted by ultrasonic treatment in volumes of about 6-8 ml each. Protein release increased with time, but enzyme activity reached a peak alter 1 rain of sonication. Maximal urogen III formation was found at 60-90 sec, then it significantly diminished, concomitantly increasing its decarboxylation products, indicating both a good liberation of the urogen decarboxylase enzyme and inactivation of isomerase and/or PBGase (data nol shown).

Properties That Rp. palustris PBGase is also a soluble enzyme was demonstrated because no significant activity was measured in the 27,000 g pellet fraction, at variance with our own findings in Euglena gracilis in which we were able to detect both a soluble and a particulate PBGase (Rossetti et al., 1986). Activity of Rp. palustris PBGase was assayed both in the presence and the absence of oxygen, neither the activity of the homogenate and the supernatant fraction nor the isomeric type of both the porphyrins formed and the PBG consumed were affected by changing the atmosphere conditions (data not shown). To study the effect of PBG concentration, experiments were carried out varying the amount of substrate between 10 and 450/~M (Fig. 2). Uroporphyrinogen formation shows a sigmoidal pattern, while PBG consumption increases linearly without reaching a plateau. So, a final substrate concentration of 177 IIM was routinely employed during PBGase assays. On the other hand, we can recall that Llambias and Baffle (1971a), Rossetti (1978) and more recently Kotler et al. (1987b) have reported that uroporphyrinogen synthesis did not follow the stoichiometric relation of 4 m o l of substrate to 1 mol of product, which instead was demonstrated by Williams et al. (1981). Employing Rp. palustris PBGase, an excess of three times in PBG consumption at low substrate concentration (up to 30/~M) was observed, followed by a two-fold excess between

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293

Rp. palustris porphobilinogenase 40 and 130/JM and finally a linear increase up to 4 4 0 # M (Fig. 2). We consider that the inhibitory effect of hydroxymethylbilane on URO-S activity reported by Battersby et al. (1983) in Euglena gracilis could be the most reasonable explanation for this great deviation in reaction stoichiometry. When the enzyme concentration effect was investigated, it was found that both porphyrin formation and PBG consumption reached their maximum at 0.8 mg protein (Fig. 3a). If we consider instead specific activity (Fig. 3b) a maxima at 0.24).4 mg was visualized for porphyrin formation, higher amounts of protein produced a dramatic fall. When PBG consumption was analysed, specific activity rapidly diminished with increasing enzyme. This effect could he attributed to some kind of protein-protein interaction, which we found to be more pronounced when working with less purified fractions. To obtain both reproducible and accurate results about 0.4rag protein were employed in the standard incubation mixture. It was found that porphyrinogen formation increased linearly with time up to 2 hr and then seemed to reach a plateau more slowly (Fig. 4). Under the same conditions, maximal PBG consumption occurred after the first hour, then no further substrate appeared to be used. These results would indicate that initially PBG was rapidly consumed to form the hydroxymethylbilane, from which uroporphyrinogen III is formed at a relatively lower but linear rate, until all the precursor is used up by the isomerase. A slight decrease in the percentage of type III with time was also observed. This time-dependent loss of cosynthase activity has already been reported (Levin, 1968; Clement et al., 1982). PBGase shows a sharp optimum pH at 7.4 either in Tris or phosphate buffer, when activity was measured in terms of porphyrins formed (Fig. 5). When

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Fig. 5. Effect of pH on Rp. palustris PBGase activity. Experimental conditions are indicated in Materials and Methods. ((3,O) 0.05M Na-phosphate buffer, (D,B) 0.05 M Tris-HCl buffer, (A,A) 0.05 M glycine~NaOH buffer. Activity expressed as porphyrin formed (©,l-L/x) and PBG consumed ( O , . , A ) .

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requires the presence of oxygen and a reducing agent for activity. The possibility that this enzyme could be related with the second maxima observed at pH 8.5 for substrate consumption (Fig. 5) and with the great deviation found in reaction stoichiometry (Fig. 2) was considered. The optimum pH for rat liver PBGoxygenase (Tomaro et al., 1973) and wheat germ (Frydman et al., 1973b), was found to be between 7 and 8; but there is a report o f a pH 8.5 for tryptophan pyrrol-oxygenase (Frydman et al., 1973a), indicating that this type of oxidase could exhibit maxima at higher pHs. Nevertheless if we also take into account that urogen III biosynthesis and PBG consumption were not affected by changing from anaerobic to aerobic conditions, it can be ruled out that PBGoxygenase is responsible for those findings. In conclusion, the reaction for porphyrinogen synthesis has a single optimum pH (7.4) whereas the existence of two different pH optima, at which PBG could bind to the enzyme, was visualized, although very likely only one (pH 7.4) is related to PBGase activity. M o l e c u l a r u't determination Rp. palustris PBGase showed an apparent mol. wt of 115,000-4-11,500 on Sephadex G-100 gel chromatography (Fig. 6, peak I), which is coincident with the PBGase mol. wt reported for avian erythrocytes (Llambias and Batlle, 1971b). A value of 50,000 + 5000, in good agreement with PBGase values reported for Euglena gracilis (Rossetti et al., 1986) and pig liver (Fumagalli et al., 1982) was calculated for peak II, where PBGase activity eluted associated with a low percentage of urogen decarboxylase ( U R O - D ) , as shown by chromatographic analysis of porphyrins formed.

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F r o m these plots apparent K m values of 50 and 1.6 × 103#M and V = 10nmol/hr and 1.1 × 103 nmol/hr were obtained for porphyrinogen formation and PBG consumption respectively. Acknowledgements This work was supported by grants from the CONICET (Argentine National Research Council), SECYT, Secretaria de Salud Publica del Ministerio de Salud Publica, Banco de la Nacion Argentina and the University of Buenos Aires. Alcira M. del C. Batlle and A. A. Juknat hold the post of Scientific Researchers in the CONICET. M. Kotler and G. Koopmann were Research Fellows of the CONICET. We wish to express our gratitude to Dr M. V. Rossetti for her kind assistance in some experiments and also to Miss H. Gasparoli and Mrs D. B. Riccillo de Aprea for their skilful technical help.

Kinetic studies

When plots of velocity against substrate concentration were analysed, it was found that measuring the rate in terms of urogen III formation, saturation curves were sigmoidal, double reciprocal plots were non-linear (Fig. 7), Eadie graphics were bell shaped and the Hill equation showed that n = 2. On the other hand, when PBG consumption was considered, velocity plots showed the classical Michaelis-Menten kinetics with n = 1 (Fig. 7).

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REFERENCES

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Rp. palustris porphobilinogenase Burton G., Fagerness P. E., Hosozawa S., Jordan P. and Scott I. (1979) 13C-NMR evidence for a new intermediate, pre-uroporphyrinogen, in the enzymic transformation of porphobilinogen into uroporphyrinogens I and III. J.C.S. Chem. Comm. 202-204. Clement R. P., Kohashi M. and Piper W. (1982) Rat hepatic uroporphyrinogen III cosynthetase: Purification, properties and inhibition by metal ions. Archs Biochem. Biophys. 214 (2), 657-667. Cohen-Bazire G., Sistrom W. R. and Stanier R. Y. (1957) Kinetic studies of pigment synthesis by non-sulfur purple bacteria. J. cell comp. Physiol. 49, 25-68. Davies R. and Neuberger A. (1973) Polypyrroles formed from porphobilinogen and amines by uroporphyrinogen synthetase of Rhodopseudomonas spheroides. Biochem. J. 133, 471 492. Frydman B., Frydman R. and Tomaro M. (1973a) Pyrroloxygenases: a new type of oxidases. Molec. cell. Biochem. 2 (2), 121-136. Frydman R., Tomaro M., Wanschelbaum A., Andersen E., Awruch J. and Frydman B. (1973b) Porphobilinogen from wheat germ: isolation, properties and products formed. Biochemistry 12 (26), 5253-5262. Fumagalli S., Rossetti M. V., Juknat A. A., Kotler M. L. and Batlle A. M. del C. (1982) Estudios sobre la PBG-asa de higado de cerdo. An. Asoc. quire, argent. 70, 375-382. Heath H. and Hoare D. S. (1959) The biosynthesis of porphyrins from porphobilinogen by Rhodopseudomonas spheroides. Biochem. J. 72, 14-22. Hoare D. S. and Heath H. 0959) The biosynthesis of porphyrins from porphobilinogen by Rhodopseudomonas spheroides. Biochem. J. 73, 679-690. Jordan P., Burton G., Nordlov H., Schneider M., Pryde L. and Scott I. (1979) Pre-uroporphyrinogen: a substrate for uroporphyrinogen III cosynthetase. J.C.S. Chem. Comm. 204-2O5. Jordan P. and Shemin D. 0973) Purification and properties of uroporphyrinogen I synthetase from Rp. spheroides. J. biol. Chem. 248, 1019-1024. Juknat A. A. (1983) Biosynthesis of porphyrinogens. Ph.D. Thesis. University of Buenos Aires, Argentina. Keilin D. and Hartree E. F. (1951) Purification of horse radish peroxidase and comparison of its properties with those of catalase and methaemoglobin. Biochem. J. 49, 88 104. Kotler M. L., Fumagalli S. A., Juknat A. A. and Batlle A. M. del C. (1987a) Porphyrin biosynthesis in Rp. palustris--VIII. Purification and properties of deaminase. Comp. Biochem. Physiol. g7B, 601-606. Kotler M. L., Fumagalli S. A., Juknat A. A. and Batlle A. M. del C. (1987b) Porphyrin biosynthesis in Rp.

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palustris--IX. PBG-deaminase. Kinetic studies. Int. J. Biochem. 19 (10), 981-985. I.~vin E. 0968) Uroporphyrinogen III cosynthetase from mouse spleen. Biochemistry 7 (1 l), 3781-3788. Levin E. (197 l) Enzymatic properties of uroporphyrinogen III cosynthetase. Biochemistry 10 (25), 4669-4675. Llambias E. and Batlle A. M. del C. (1971a) Studies on the porphobilinogen deaminase-uroporphyrinogen cosynthetase system of cultured soybean cells. Biochem. J. 121, 327-340. Llambias E. and Batlle A. M. del C. (1971b) Porphyrin biosynthesis--VIII. Avian erythrocyte porphobilinogen deaminase-uroporphyrinogen cosynthetase, its purification, properties and the separation of its components. Biochim. biophys. Acta 227, 180-191. Lockwood W. H. and Rimington C. (1957) Purification of an enzyme converting porphobilinogen to uroporphyrinogen. Biochem. J. 67, 8-11. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Moore D. and Labbe R. (1964) Assays for ALA and PBG determination. Clin. Chem. 10, I105-I109. Rimington C. (1960) Spectral absorption coefficients of some porphyrins in the Soret band region. Biochem. J. 75, 620~23. Rossetti M. V. (1978) Enzymic cyclotetramerization of PBG. Its mechanism. Ph.D. Thesis. University of Buenos Aires, Argentina. Rossetti M. V., Lombardo M. E., Juknat de Geralnik A. A., Araujo L. S. and Batlle A. M. d¢l C. (1986) Porphyrin biosynthesis in Euglena gracilis--V. Soluble and particulate PBG-ase. Comp. Biochem. Physiol. 85B, 451-458. Rossetti M. V., Araujo L. S., Lombardo M. E., Correa Garcia S. and Batlle A. M. del C. (1987) Porphyrin biosynthesis in Euglena gracilis--VI. The effect of light and growth conditions on PBG-ase activity and further properties. Comp. Biochem. Physiol. g/B, 593-600. Sancovich H. A., Batlle A. M. del C. and Grinstein M. (1969) Porphyrin biosynthesis--VI. Separation and purification of PBG-deaminase and uroporphyrinogen isomerase from cow liver. PBG-ase an allosteric enzyme. Biochim. biophys. Acta 191, 130-143. Tomaro M. L., Frydman R. and Frydman B. (1973) Porphobilinogen oxygenase from rat liver: induction, isolation and properties. Biochemistry 12 (26), 5263-5268. Williams D. C., Morgan G. S., McDonald E. and Battersby A. R. 0981) Purification of porphobilinogen deaminase from Euglena gracilis and studies of its kinetics. Biochem. J. 193, 301-310.